Evaluation dopamine receptors in Parkinson's

描述（由申请人提供）：帕金森病（PD）由黑质纹状体多巴胺能神经元变性引起。该过程最终导致多巴胺介导的纹状体信号传导的进行性减少，其表现为以运动迟缓、僵硬、震颤和步态异常为特征的临床综合征。PD的传统疗法旨在通过施用多巴胺前体左旋多巴来恢复纹状体中的多巴胺水平。对于晚期疾病，左旋多巴会导致运动障碍和运动活动的明显波动（“开关效应”）。减轻这些副作用一直是一个重大的挑战，并促使寻找替代策略，可以提供更稳定的多巴胺能信号传导水平。先前未探索的恢复纹状体多巴胺能活性并同时潜在地避免长期L-多巴施用的后果的选择是通过引入组成性活性多巴胺受体。PI实验室在通过引入激活点突变产生具有配体非依赖性（或组成性）活性的受体方面具有丰富的经验。这些受体具有即使在没有多巴胺和/或多巴胺能激动剂药物的情况下维持多巴胺能信号传导的潜力。本申请的前提是，组成型活性多巴胺受体可以使用体外测定法鉴定并在大鼠纹状体中表达，以在延长的时间间隔内增强多巴胺能信号传导。具体目标1的目的是在体外产生并表征一系列组成型活性多巴胺1和多巴胺2受体。使用重组腺相关病毒，将在大鼠中探索组成型活性多巴胺受体纹状体过度表达的功能后果（具体目标2）。单侧病毒给药后的循环行为将用作构建体活性的指标。使用的方法将包括分子（组成型活性突变受体的产生，重组蛋白的表达），药理学（放射性配体结合，第二信使信号传导测定）和行为方法（评估盘旋行为）。这些实验将为纹状体中多巴胺能受体的作用提供额外的见解，并可能为开发帕金森病的新治疗方案迈出第一步。

DESCRIPTION (provided by applicant): Parkinson's disease (PD) results from the degeneration of nigrostriatal dopaminergic neurons. This process ultimately leads to a progressive decrease in dopamine mediated striatal signaling which manifests as a clinical syndrome characterized by bradykinesia, rigidity, tremor, and gait abnormalities. Traditional therapy for PD has aimed at restoring dopamine levels in the striatum through administration of the dopamine precursor, L-dopa. With advanced disease, L-dopa leads to dyskinesias and periods of marked fluctuation in motor activity ('on-off effect'). Alleviation of these side effects has been a major challenge and has prompted a search for alternative strategies which can provide a more stable level of dopaminergic signaling. A previously unexplored option to restore striatal dopaminergic activity and at the same time to potentially avoid the consequences of long term L-dopa administration, is through the introduction of constitutively active dopamine receptors. The laboratory of the PI has extensive experience in generating receptors with ligand independent (or constitutive) activity through the introduction of activating point mutations. These receptors have the potential to maintain dopaminergic signaling even in the absence of dopamine and/or dopaminergic agonist drugs. The premise of this application is that constitutively active dopamine receptors can be identified using in vitro assays and expressed in the striatum of rats to enhance dopaminergic signaling over an extended time interval. The objective of Specific Aim 1 is to generate and pharmacologically characterize in vitro a series of constitutively active dopamine 1 and dopamine 2 receptors. Using recombinant adeno-associated virus, the functional consequences of striatal overexpression of constitutively active dopamine receptors will be explored in rats (Specific Aim 2). Circling behavior after unilateral viral administration will be used as an index of construct activity. The methodologies utilized will include molecular (generation of constitutively active mutant receptors, expression of recombinant proteins), pharmacologic (radioligand binding, second messenger signaling assays), and behavioral approaches (assessment of circling behavior). These experiments will provide additional insight into the role of dopaminergic receptors in the striatum as well as potentially take the first steps toward the development of a new therapeutic option for Parkinson's disease.